Available online at Department of Animal Reproduction, FMVZ-USP, São Paulo-SP, Brazil b

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1 Available online at Theriogenology 78 (2012) Effects of two estradiol esters (benzoate and cypionate) on the induction of synchronized ovulations in Bos indicus cows submitted to a timed artificial insemination protocol J.N.S. Sales a,f, *, J.B.P. Carvalho b, G.A. Crepaldi c, R.S. Cipriano d, J.O. Jacomini a, J.R.G. Maio c, J.C. Souza e, G.P. Nogueira d, P.S. Baruselli a a Department of Animal Reproduction, FMVZ-USP, São Paulo-SP, Brazil b APTA-Vale do Paraíba Regional Center, Pindamonhangaba, SP, Brazil c Ouro Fino Saúde Animal, Ribeirão Preto, SP, Brazil d Department of Support, Animal Production and Health - Unesp, Araçatuba, SP, Brazil e Department of Animal Science, UFLA, Lavras, MG, Brazil f Center of Veterinary Science, UFPB, Areia, PB, Brazil Received 17 December 2011; received in revised form 26 February 2012; accepted 26 February 2012 Abstract The effects of estradiol benzoate (EB) and estradiol cypionate (EC) on induction of ovulation after a synchronized LH surge and on fertility of Bos indicus females submitted to timed AI (TAI) were evaluated. In Experiment 1, ovariectomized Nelore heifers were used to evaluate the effect of EB (n 5) and EC (n 5) on the circulating LH profile. The LH surge timing (19.6 and 50.5 h; P 0.001), magnitude (20.5 and 9.4 ng/ml; P 0.005), duration (8.6 and 16.5 h; P 0.001), and area under the LH curve (158.6 and ng/ml; P 0.01) differed between the EB and EC treatments, respectively. In Experiment 2 (follicular responses; n 60) and 3 (pregnancy per AI; P/AI; n 953) suckled Bos indicus beef cows submitted to an estradiol/progesterone-based synchronization protocol were assigned to receive one of two treatments to induce synchronized ovulation: 1 mg of EB im 24 h after progesterone (P4) device removal or 1 mg of EC im at P4 device removal. There was no difference (P 0.05) between EB and EC treatments on follicular responses (maximum diameter of the ovulatory follicle, 13.1 vs mm; interval from progesterone device removal to ovulation, 70.2 vs h; and ovulation rate, 77.8 vs. 82.8%, respectively). In addition, P/AI was similar (P 0.22) between the cows treated with EB (57.5%; 277/482) and EC (61.8%; 291/471). In conclusion, despite pharmacologic differences, both esters of estradiol administered either at P4 device removal (EC) or 24 h later (EB) were effective in inducing an LH surge which resulted in synchronized ovulations and similar P/AI in suckled Bos indicus beef cows submitted to TAI Elsevier Inc. Open access under the Elsevier OA license. Keywords: Bovine; Ovarian response; Estradiol; Tai; LH; Synchronization 1. Introduction * Corresponding author. Tel.: address: znlogan@yahoo.com.br (J.N.S. Sales). It is currently possible to efficiently synchronize ovarian follicular growth and induce ovulation of a dominant follicle at a known moment, allowing insemination of beef zebu females on a predetermined day and hour, without the need to detect estrus [1]. Timed X 2012 Elsevier Inc. Open access under the Elsevier OA license.

2 J.N.S. Sales et al. / Theriogenology 78 (2012) artificial insemination (TAI) may be employed either in cyclic or anestrous cows and started independent of estrous cycle phase [2]. Recently, many ovulation synchronization protocols have been studied in zebu cows [3 5]. These protocols are based on synchronizing emergence of the wave of follicular growth by simultaneous administration of progesterone and estradiol [6,7]. At the end of a protocol, it is necessary to reduce circulating progesterone concentrations by removal of the progesterone device (exogenous source) and administration of prostaglandin to promote CL regression (endogenous source), so that, ovulation may occur. Lastly, it is necessary to promote the final growth of the follicle and to induce a synchronized ovulation, which allows insemination at a predetermined moment [2]. Adequate results have been achieved by TAI ( 50%) with protocols employing estradiol benzoate (EB) as the ovulation inducing agent in Bos taurus [8,9] and in Bos indicus [1,4] beef cows. However, it is necessary to handle cows at least four times to perform TAI (Day 0, EB P4 device insertion; Day 8, PGF P4 device removal; Day 9, EB; and Day 10, TAI [10]). It is known that estradiol esters are capable of inducing ovulation in beef cows, however, their pharmacokinetics differ. In some studies, EB had a shorter half-life and induced an earlier LH peak compared to estradiol cypionate (EC) [11,12]. Thus, based on the distinct pharmacokinetics of estradiol esters and aiming to reduce TAI-related cow handling, three studies were conducted to evaluate the effect of EB and EC on induction of ovulation of suckled beef cows submitted to TAI. The objective of Experiment 1 was to evaluate the circulating LH-release profile after giving EB and EC administration to ovariectomized Bos indicus heifers. Experiment 2 aimed to evaluate the effect of EC (treated at P4 device removal) or EB (treated 24 h post P4 device removal) on ovarian responses of Nelore cows (Bos indicus) submitted to a TAI protocol. Experiment 3 aimed to evaluate the effect of EC and EB, as ovulation inducers, on the pregnancy rate of Nelore cows (Bos indicus) submitted to TAI. The hypothesis was that EC administration (at device removal) decreases cow handling without affecting fertility of beef cows submitted to TAI programs, compared to EB given 24 h after P4 device removal. 2. Materials and methods 2.1. Experiment 1: circulating LH profiles in ovariectomized Nelore heifers Animals and handling Experiment 1 was conducted on the research farm of the University of the State of Sao Paulo (UNESP) in Araçatuba, SP, Brazil. Nelore heifers, mo old, weighing kg had their ovaries removed (via laparotomy) to eliminate endogenous estradiol. Heifers were maintained on Brachiaria brizantha pastures and given mineralized-salt and free access to water Experimental design Ovariectomized heifers (n 10) were synchronized using an estradiol/progesterone-based TAI protocol 1. Heifers received an intravaginal device containing 1 g of P4 (Sincrogest, Ourofino Agronegocio, Sao Paulo, Brazil) plus 2 mg of estradiol benzoate im (EB; Sincrodiol, Ourofino Agronegocio, Sao Paulo, Brazil). Eight days later, the progesterone device was removed and heifers received 500 g PGF 2 im (sodium cloprostenol; Sincrocio, Ourofino Agronegocio, Sao Paulo, Brazil). Concurrently, EC-Group heifers (n 5) received 1 mg of estradiol cypionate im (ECP, Pfizer, Sao Paulo, SP, Brazil) and EB-Group heifers (n 5) received 1 mg EB im (Sincrodiol, Ourofino Agronegocio, Sao Paulo, SP, Brazil) Blood sampling and LH assay Jugular veins were cannulated 1 day before onset of treatments with the two estradiol esters. Blood samples were collected in evacuated tubes every 3 h after initiation of treatments (total of 72 h). Within 4 h after collection, samples were refrigerated (4 C) and then centrifuged (3000g for 15 min) and stored at 20 C until assayed for LH. Plasma concentrations of LH were determined by double antibody radioimmunoassay, as previously described [13]. The intraassay and interassay CVs were 10.3 and 13.1%, respectively. Assay sensitivity was 0.02 ng/ml Experiment 2: ovarian responses in Nelore cows Animals and handling procedures The second experiment was conducted at the State Research Farm (APTA - Pindamonhagaba Regional Center), in Pindamonhagaba, Sao Paulo, Brazil. Sixty suckled multiparous Nelore cows scoring on a (1-thin to 5-obese) body condition score (BCS; [14]) scale. All cows were maintained on Brachiaria brizantha pastures and given mineralized-salt and free access to water Experimental design All cows were synchronized using an estradiol/progesterone-based TAI protocol 1. Hormonal treatments started between 30 and 60 days postpartum. Cows received an intravaginal device containing 1 g of P4 (Sincrogest, Ourofino Agronegocio, Sao Paulo, Brazil) plus an administration of 2 mg of estradiol benzoate im

3 512 J.N.S. Sales et al. / Theriogenology 78 (2012) Fig. 1. Experimental design (P4, progesterone; EB, estradiol benzoate; EC, estradiol cypionate; ecg, equine chorionic gonadotropin; TAI, Timed AI). (EB; Sincrodiol, Ourofino Agronegocio). Eight days later, the device was removed and 500 g PGF 2 (cloprostenol sodium; Sincrocio, Ourofino Agronegocio) and 300 IU of equine chorionic gonadotropin (ecg; Folligon, Intervet-Schering-Plough, Boxmeer, Netherlands) administered im. At P4-device removal, EC- Group cows (n 30) received 1 mg of estradiol cypionate im (ECP, Pfizer Animal Health, Sao Paulo, SP, Brazil) and EB-Group cows (n 30) received 1 mg EB im (Sincrodiol, Ourofino Agronegocio) 24 h after P4- device removal (Fig. 1). At the beginning of the estrous synchronization procedures (day 0), ultrasonographic examinations were performed (USA; Chison 600 VET, 5 MHz linear transducer, China) to evaluate ovarian status (presence of CL and ovarian follicles 8 or 8 mm [1]). Moreover, ultrasonographic evaluations were performed every 12 h from P4-device removal to detection of ovulation. The moment of ovulation was defined as the time of disappearance of a previously identified dominant follicle (DF) from one ultrasound examination to the next, minus 6 h Experiment 3: pregnancy per AI in Nelore cows (Bos indicus) Animals and management The experiment was conducted on two commercial farms located in northwestern Brazil, from January to March A total of 953 multiparous suckled Nelore (Bos indicus) cows, 30 to 60 days postpartum and average BCS of , were used [14]. All cows were maintained on Brachiaria brizantha pastures and given mineralized-salt and free access to water Experimental design Postpartum suckled cows were allocated into treatment groups according to calving date. All cows were synchronized using an estradiol/progesterone-based TAI protocol and treatments similar to those described in Experiment 2. In this experiment, TAI was performed 48 to 52 h after P4 device removal. Frozen semen straws from single ejaculates of each of the three sires were homogeneously distributed between experimental groups. All inseminations were performed by the same technician who had no previous knowledge of cow treatment allocation. Pregnancy examinations were conducted by transrectal ultrasonography (Chison D600VET, Chison Medical Imaging, Co, China) 30 days after AI. The experimental design is shown (Fig. 1) Statistical analysis Statistical analyses were performed using Statistical Analysis System for Windows [15]. In Experiment 1, LH surge was defined as an increase in LH 2SD above the overall within-cow mean of LH concentrations. The magnitude of the LH surge (MLH) was defined as the difference between the maximum concentrations observed during the surge and baseline. Timing of LH surge (TLH) was defined as the moment in which the LH surge occurred. Duration of the LH surge (DLH) was determined as the time interval from the onset of the LH surge (baseline concentration before the increase in LH concentration induced by estradiol) to its first return to baseline concentrations. Area under the curve (AUC) of LH release was calculated by the trapezoid method. In Experiment 2, the variables evaluated were diameter of the ovulatory follicle, time from P4-device removal to ovulation, and

4 J.N.S. Sales et al. / Theriogenology 78 (2012) ovulation rate. Continuous data were tested for normality of the residues and analyzed by the UNIVARIATE procedure (transformed when necessary) and subjected to Bartlett s test to assess homogeneity of variances. The GLM procedure with Tukey adjustment was used to determine significant differences among groups. All values are expressed as mean SEM. In Experiment 3, the variables initially included in the models were treatment (EB and EC), farm and BCS at the first day of the synchronization protocol, and their interactions. Data were analyzed by a multivariate logistic regression using the LOGISTIC procedure of SAS. Variables were removed by backward elimination, based on the Wald statistics criterion when P Variables included in the final model for analysis of P/AI were treatment, BCS and farm. Adjusted odds ratio (AOR) and 95% confidence interval (CI) were generated during the logistic regression. Results are presented as proportions and AOR. The P/AI was analyzed using the GLIMMIX procedure of SAS. 3. Results 3.1. Experiment 1: circulating LH profile in ovariectomized Nelore heifers Table 1 Plasma LH profile in ovariectomized Nelore heifers treated with EC or benzoate (EB). EB EC P No. animals 5 5 Magnitude of LH surge (ng/ml) Time of LH surge (h) Duration of LH surge (h) Area under LH curve (ng/ml/72 h) Protocol: day 0 1 intravaginal device of P4; 2 mg of EB; Day 8 - progesterone device was removed; 500 g PGF 2 ; treatments (EC Group 1 mg of estradiol cypionate or EB Group 1 mg estradiol benzoate). The LH release profile was different between ECand EB-Group heifers (Fig. 2). The TLH, MLH, DLH and AUC for the EC and EB treatments are summarized (Table 1). Onset of the LH peak differed (P 0.001) between EC and EB groups, such that in the latter LH peaked 30.9 h earlier than in the former. Additionally, LH peak magnitude was greater (P 0.005) for EB heifers. Moreover, heifers receiving EB as the ovulation inducing agent had shorter (P 0.001) peak duration and smaller (P 0.01) area under the curve for LH concentrations. Finally, LH peak dispersion was smaller (P 0.01) in Group-EB compared to that of EC-Group heifers Experiment 2: ovarian responses in Nelore cows Of the 60 cows initially used in this experiment, four (6.7%) did not respond (absence of a DF 6 mmat P4-device removal) to the TAI protocol and were therefore removed from the study. Follicular response data are shown (Table 2). Maximum diameters of the DF (P 0.12), and of the ovulatory follicle (P 0.11) and interval from P4-device removal to ovulation did not differ between cows that received EB 24 h after P4- device removal and those receiving EC as described. Ovulation rate after the TAI protocol was similar between the EB (77.8%; 21/27) and the EC groups Fig. 2. Mean SEM effect of treatment with EC or EB on circulating LH concentrations in Nelore cows. Table 2 Effects of EB - estradiol benzoate or EC - estradiol cypionate, as ovulation inducers in suckled Nelore cows submitted to progesterone-based TAI protocols, on ovarian responses. EB EC P No. animals Diameter of DF on Day (mm) Maximum diameter of the DF (mm) Maximum diameter of the OF (mm) Interval P4 device removal to ovulation (h) Ovulation rate (%) 77.8 (21/27) 82.8 (24/29) 0.82 Double ovulation 4.8 (1/21) 4.2 (1/24) 0.89 DF, dominant follicle; OF, ovulatory follicle.

5 514 J.N.S. Sales et al. / Theriogenology 78 (2012) Fig. 3. Distribution of the time of ovulation (h) after P4 device removal in Nelore cows (Bos indicus) submitted to TAI according to EC treatment at P4 device removal (handled three times; n 29) or EB 24 h after (handled four times; n 27). (82.8%, 24/29). Similarly, there was no difference in double ovulation rates (EB 4.8%; 1/21 and EC 4.2%; 1/24). Moreover, the variability of ovulation was also similar (P 0.59) between treatments (Fig. 3) Experiment 3: pregnancy per AI in Nelore cows (Bos indicus) There was no interaction between ovulation inducer and explanatory variables, such as BCS (P 0.96) and farm (P 0.21) on P/AI. There was no significant effect of ovulation inducer (EB and EC; P 0.22) and farm (P 0.15) on P/AI (Table 3). However, increased BCS on the first day of the synchronization protocol was associated with increases in P/AI (Table 3). 4. Discussion To our knowledge, this is the first report studying the effects of distinct ovulation inducers in estradiol and progesterone based protocols on LH profiles, follicular responses and fertility of Bos indicus females submitted to TAI. In the present study, despite differences between the LH releases characteristics of the two ovulation inducers, ovarian response and fertility of Bos indicus cows were similar. These results were reached because of the 24 h earlier EC administration used in the ovulation synchronization protocol. The experimental hypothesis was confirmed, since fertility between groups was similar. The main advantage of EC is the reduced need to handle these animals to perform the TAI. LH peaks occurred 19.6 and 50.5 h after the administration of EB and EC, respectively. These results were consistent with previous studies in which the administration of EB to beef cows (Bos taurus) or EC to crossbred beef heifers promoted LH surges at approximately 20.4 [12] or 54.6 h [11] after treatment, respectively. This is result of the distinct pharmacokinetics of the estradiol esters used. The EC is formed by esterification of estradiol by cyclepentano propionic acid, resulting in low solubility in water and consequent slower release from the administration site and prolonged biological activity compared to estradiol benzoate [16]. In addition to LH peak anticipation, differences in the pharmacokinetics of the estradiol esters are responsible for the greater LH release and shorter LH peak duration of animals treated with estradiol benzoate. The recognition of these differences is important to determine the appropriate time to administer the ovulation inducer in TAI protocols in which estradiol and progesterone are used. Despite the greater LH peak synchronization of animals receiving EB as the ovulation inducer, ovulation synchronization between the two experimental groups (EB and EC) was similar. Presently, progesterone and estradiol based TAI protocols are highly efficient in synchronizing ovulation in Bos indicus cows [17,18]. In the present study, 80% of the cows responded to the synchronization protocol and in only 6.7% a DF was not detected at P4-device removal. Other authors reported that 4.7% [19] and 6.3% [20] of females synchronized with estradiol and progesterone based protocols did not exhibit a new wave of follicular growth. Another important characteristic of TAI protocols is the ovulatory follicle diameter (OFD), because it is directly related to CL size during the subsequent diestrus [21]. The CL diameter is highly correlated to the progesterone it synthesizes [22] which is essential for Table 3 Risk factors for P/AI following two estradiol/progesterone-based synchronization protocols in suckled Bos indicus cows. P Variable P/AI, % (n/n) Adjusted OR (95% CI) a Ovulation inducer EB 57.5 (277/482) Referent b 0.22 EC 61.8 (291/471) 1.18 ( ) Farm A 58.7 (229/390) Referent b 0.15 B 60.2 (339/563) 1.24 ( ) BCS category Low ( ) 54.9 (218/397) Referent b 0.01 Moderate to high ( 2.5) 63.0 (350/556) 1.39 ( ) a OR, odds ratio; CI, confidence interval. b Referent, reference group for adjusted risk ratio.

6 J.N.S. Sales et al. / Theriogenology 78 (2012) post-fertilization embryonic development [23]. In the present study, OFD did not differ between ovulation inducers or between treatments and diameter of the ovulatory follicle was 13 mm at TAI. Satisfactory pregnancy rates were observed in cows with OF diameters 13 mm at TAI [24]. Besides their efficiency in synchronizing the LH peak, both estradiol esters induced high ovulation rates ( 80%), approximately 70 h after P4-device removal. Ovulation synchronization allows cows to be inseminated at a predetermined time point, thereby avoiding the need for estrus detection [1]. Ovulation synchronization is important in situations in which the flexibility in relation to the time of AI is low, as in cows inseminated with sex-sorted semen [18]. The moment of ovulation in relation to the P4- device removal was similar between estradiol esters because of the 24-h delay on the EB administration, since the LH peak in this group occurred 30 h earlier than in EC group. This delay allowed a similar final growth of the ovulatory follicle between cows receiving either EB or EC. In a previous study, EB administration at progesterone device removal resulted in smaller ovulatory follicle diameters and lower pregnancy rates in cows inseminated 54 h after P4-device removal [17]. Fertility was not influenced by estradiol esters in this study. Similar results were observed by Meneghetti, et al. [25] in which no pregnancy rate differences were detected between cows treated with EB (50.8%) 24 h after P4-device removal or EC (51.9%) at P4-device removal. The estradiol esters studied had similar efficacy in inducing ovulation and fertility for TAI in beef cows. Body condition scores lower than 2.5 (1 5 scale) led to lower TAI pregnancy rates in beef cows [2]. Inthe present study, the risk for P/AI was higher in cows of moderate to high BCS in both treatments. In various studies with beef cows, it was reported that low BCS at the beginning of TAI synchronization protocols may reduce fertility [4,25,26]. Thus, management techniques aiming at maintain or improving BCS before and, mainly, after parturition are important to guarantee the future fertility of cattle herds. In conclusion, despite pharmacologic differences between the two ovulation inducing agents studied (EB and EC), both were effective in inducing an LH peak which resulted in synchronized ovulations, adequate fertility in suckled Bos indicus beef cows submitted to an estradiol/progesterone-based synchronization protocol. However, the use of EC allows for reduction on the number times cows must be handled without reduced fertility. This is also a desirable characteristic for increasing the adoption and spread of TAI. Acknowledgments The authors are thankful to DIMAFE Agropecuária (Jacareuna Farm - são Felix do Araguaia, MT, Brazil), APTA-Vale do Paraíba Regional Center (Pindamonhangaba, SP, Brazil) and Department of Support, Animal Production and Health - UNESP (Araçatuba, SP, Brazil) for allowing the use of their animals and facilities for this study. This research was supported by Ourofino Agronegócio (Cravinhos, SP, Brazil), RG Genética Avançada (Água Boa, MS, Brazil) and CAPES. References [1] Baruselli PS, Reis EL, Marques MO, Nasser LF, Bo GA. The use of hormonal treatments to improve reproductive performance of anestrous beef cattle in tropical climates. Anim Reprod Sci 2004;82/83: [2] Bó GA, Baruselli PS, Martínez MF. Pattern and manipulation of follicular development in Bos indicus cattle. Anim Reprod Sci 2003;78: [3] Baruselli PS, Marques MO, Carvalho NAT, Madureira EH, Campos Filho EP. Effect of different treatments for timed artificial insemination on the reproductive efficiency in lactating beef cows. Rev Bras Reprod Anim 2002;26: [4] Sá Filho OG, Meneghetti M, Peres RFG, Lamb GC, Vasconcelos JLM. Fixed-time artificial insemination with estradiol and progesterone for Bos indicus cows II: Strategies and factors affecting fertility. Theriogenology 2009;72: [5] Sales JN, Crepaldi GA, Girotto RW, Souza AH, Baruselli PS. Fixed-time AI protocols replacing ecg with a single dose of FSH were less effective in stimulating follicular growth, ovulation, and fertility in suckled-anestrus Nelore beef cows. Anim Reprod Sci 2011;124:12 8. [6] Bó GA, Baruselli PS, Moreno D, Cutaia L, Caccia M, Tríbulo R, et al. The control of follicular wave development for selfappointed embryo transfer programs in cattle. Theriogenology 2002;57: [7] Carvalho JB, Carvalho NA, Reis EL, Nichi M, Souza AH, Baruselli PS. 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